**Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.

**Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.

-

**Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.

+

**Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.

*You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

*You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

### Undiluted cells in this experiment are actually not very concentrated. Glycerol water of volume 1/500th of the culture volume was mixed back in. [[User:Janet B. Matsen|Janet]] has subsequently (8/2013) learned that more concentrated is much better, and is now routinely getting lawns from gibson assembly transformations.

*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells| this page]].

+

* [[User:Janet B. Matsen|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.

Chemically Competent E. Coli

Notes:

You need fresh cells.

Often people inoculate a few mL of the culture for overnight growth, then use 200 uL to inoculate the ~50 mL of culture they will make competent.

Some people in our lab believe you want to start with a fresh plate and don't even use one that is a few days old. Other people (Mila) are mostly concerned about the OD being right at the time of harvest.

You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

Undiluted cells in this experiment are actually not very concentrated. Glycerol water of volume 1/500th of the culture volume was mixed back in. Janet has subsequently (8/2013) learned that more concentrated is much better, and is now routinely getting lawns from gibson assembly transformations.

Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See this page.

[[User:Janet B. Matsen|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.